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DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: derekb.lowe@gmail.com Twitter: Dereklowe

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February 27, 2013

Not What It Says On the Label, Though

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Posted by Derek

The topic of compound purity has come up here before, as well it should. Every experienced medicinal chemist knows that when you have an interesting new hit compound, that one of the first things to do is go back and make sure that it really is what it says on the label. Re-order it from the archive (in both powder and DMSO stock), re-order it if it's from a commercial source, and run it through the LC/MS and the NMR. (And as one of those links above says, if you have any thought that metal reagents were used to make the compound, check for those, too - they can be transparent to LC and NMR).

So when you do this, how many compounds flunk? Here are some interesting statistics from the folks at Emerald:

Recently, we selected a random set of commercial fragment compounds for analysis, and closely examined those that failed to better understand the reasons behind it. The most common reason for QC failure was insolubility (47%), followed by degradation or impurities (39%), and then spectral mismatch (17%) [Note: Compounds can acquire multiple QC designations, hence total incidences > 100% ]. Less than 4% of all compounds assayed failed due to solvent peak overlap or lack of non-exchangeable protons, both requirements for NMR screening. Failure rates were as high as 33% per individual vendor, with an overall average of 16%. . .

I very much wish that they'd identified that 33% failure rate vendor. But overall, they're suggesting that of 10 to 15% compounds will wipe out, regardless of source. Now, you may not feel that solubility is a key criterion for your work, because you're not doing NMR assays. (That's one that will only get worse as you move out of fragment-sized space, too). But that "degradation or impurities" category is still pretty significant. What are your estimates for commercial-crap-in-a-vial rates?

Comments (11) + TrackBacks (0) | Category: Chemical News | Drug Assays


COMMENTS

1. alig on February 27, 2013 10:45 AM writes...

More importantly, who is vendor X with its 0% fail rate. And does it have any relationship to Speed Racer.

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2. Anonymous on February 27, 2013 11:03 AM writes...

I have worked on QC projects before, both for starting materials and screening compounds. For the SM QC, many were too low MW for LC-MS, so we used both GC-MS and NMR. In those cases, we did often find that commercial chemicals were often not up to the spec on the label; thus the 99.5% 4-fluorobenzylamine was really 95% pure. As well, we occasionally found completely wrong compounds in a commercial source, incorrect isomers or totally wrong compound (only once or twice though). But of about 2000 chemicals tested as SMs, over 80% were an easy pass, even at a spec of 90% purity (some were only slightly less, many were only labelled 95-96% pure and some of those looked below 90%.) We also found that some compounds degraded much faster than we expected. We expected to see aldehydes, acid chlorides, isocyanates, and other highly reactive compounds degrade with time. But we saw some amines, especially benzyl amines, eg, difluorobenzylamines, has many impurities develop within a short time of passing QC.

In commercial compounds, the numbers depended on the source, much more. Compounds from reputable sources, like Maybridge were mostly good, over 90% correct and pure (some failed due to minor impurity), few were grossly impure or wrong.

But in many outsourced sets, libraries of "pure compounds" from some vendors, and compounds from smaller companies we saw huge variations in amount (some vials of "5 mg" were 1 mg or less), purity (many were far below a spec of 85% pure), and identity. I think some of that had to do with compounds that were transferred as solutions and then dried back down (they often had more degradation and impurities). Some of those companies no longer exist.

But in some cases, we determined that the QC assay was not relevant to the compound. Many chemists (both analytical and synthetic) do not understand that not all compounds absorb UV at the same level (Beers Law is only relevant for the same compound, not mixtures of compounds) and in fact, some organic compounds do not absorb UV (many steroids, aliphatics, smaller molecules) or ionize under LC-MS conditions. So a QC project that attempts to test all compounds by LC-MS, without looking at the structures or doing a backup of NMR for the compounds that failed LC-MS will likely show a higher failure rate than is real.

We tested that with 30 analytically pure (fresh USP grade or better) drug compounds that were then carefully dissolved and tested in GC-MS, LC-MS, NMR, ELSD, and CLND. We found that the purity, identity, and amount estimated (quantitation) were all over the board, although about 90% of the LC-MS tests confirmed the identity and purity, the estimated concentration of the compound was often off by an order of magnitude, thus any impurity could be very hard to quantify accurately. The NMR identity, purity, and concentration data were usually within 5-10%, but required very careful prep work, lots of d-solvents, and skilled analytical interpretation. So the current reliance on automated LC-MS QC may create many false positives and negatives. Sorry, I don't have the raw data, that was an internal publication only.

Lastly, don't forget the salt counterions in your samples. Many don't show up in some or any of these assays. If you have NO2-, NO3-, TFA-, ClCH2COO-, Br-, F-, etc in your compound, these can seriously mess up many assays. We had a team of scientist chasing after a great cardiovascular drug lead for months that turned out to be simply contaminated with nitrite/nitrate.

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3. Ben Zene on February 27, 2013 12:01 PM writes...

Yeah but people will keep saving the $$$$ by buying cheap stuff from vendors who don't characterize products adequately. Particularly when buying individual tool compounds always get a CoA and don't be shy about asking for copies of the original analytical data too. And ask suppliers for their microanalysis data in instances when a salt or metal contamination could be present. This won't be completely foolproof but it will be far better than just relying on RLS (reading the label spectroscopy).

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4. Tristan on February 27, 2013 8:20 PM writes...

Last year I purchased a compound, purportedly a small biological dipeptide (histamine-(beta-alanine), from a company whose name I shall not mention. I was rather surprised when the RP-HPLC trace of my reaction mixture revealed an enormous, late-eluting peak. I'm fairly certain that what they sent me was the remains of the fmoc protecting group used in synthesis...

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5. Trollumination on February 27, 2013 9:43 PM writes...

A certain vendor of peptides once sold my lab several 96-well plates of peptides which were all consistently and entirely missing the first residue. I suspect pump priming error or something like.

But the peptide industry is full of snakes - some have been known to bulk up a very low or even nonexistent yield by addition of buffer salts.


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6. newnickname on February 28, 2013 8:11 AM writes...

I ordered and received a difficult-to-make catalog small organic. It was the right color but, surprisingly, a granular solid. The NMR looked VERY good but it was not very soluble. I ran my reaction and the yield was abysmal. I didn't test the solid, but I'm pretty sure they sent me the sodium sulfate covered with a small amount of really clean compound. They sent me replacement bottles that were all the same. The technician who made it must have poured the filtrate with the product into the waste can and kept the Na2SO4!

(The other day I posted favorably to the Blog Syn - Baran topic about those "with ordinary skill in the art". This would be an exception to that position.)

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7. Todd on February 28, 2013 1:14 PM writes...

And this is why you characterize anything that gets brought in. I know it's only worse in the biology labs. You guys are lucky.

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8. Magpie on February 28, 2013 2:12 PM writes...

@6. We must have ordered from the same company....
I recently ordered 3-amino-4-bromopyridine (from a smallish vendor) that twice failed in a run-of-the-mill synthesis. After the second fail, I looked into its reported physical properties via Reaxys and was quite dismayed that what is supposed to be a brown oil was in fact a hard insoluble red solid. I took it up into MeOH, filtered away plenty of insolubles, and ran it on the LCMS. While a compound with the right m/z showed up, it was very impure (and that, of course, was just the soluble portion). I pulled up the CofA from their website and it was confirmed as a 98% pure brown oil. I called and spoke to the technician who ran the CofA and he suggested the compound solidified on standing. I countered that if that was the case, it would have come as one solid mass in the bottle, and not a powder with chunks. In any event, I took the NMR and was nonplussed when (amongst all of the garbage peaks) the compound's peaks were not to be found.
I called them back and insisted they refund the money.
When many hours of research time are wasted in this way, plus several hundred dollars for a few grams, you end up deleting the company from your vendor list.

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9. Dejan on March 1, 2013 5:28 AM writes...

Once, my colleague repeatedly failed to perform liquid-liquid extraction using n-BuOH (not sure about the exact manufacturer, but it was from Europe). Upon GC-MS analysis, it turned out it was (not "contained"..."was"!) a mixture of toluene and diacetone alcohol.

Also, once we got a bottle of "Methanol, C2H5OH".

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10. Secondaire on March 1, 2013 10:26 PM writes...

When I was in graduate school, I had a colleague who set out to make collaborator's lead compound so we could confirm their assay results independently. She ordered one of the starting materials, a small, hard-to-make polysubstituted aromatic. Repeatedly, the assay results were absolute rubbish. Finally, she got fed up and sent her compound for X-ray crystallography. The supplier of her starting material had sent her the wrong isomer of the thing and apparently they were practically indistinguishable by NMR. It took about six months to figure this mess out.

Had a similar "wrong isomer, you lose" situation in the current group. Supplier said "exo"; colleague proved extensively by 2D-NMR that it was endo. Supplier was subsequently read the riot act.

Permalink to Comment

11. ChristianPFC on March 4, 2013 5:01 AM writes...

I once had a bottle (Benzyl thiol, don't remember the manufacturer) that had the wrong molecular weight on the label. The number was about 10% higher than the real. Fortunately, I was using 20% excess, so I still had some excess left, so no harm was done.

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